4.2. In vitro human cell cultures

The cells in study I were human fibroblasts taken from healthy palatal gingiva and osteoblasts taken from alveolar bone. The histochemical analysis of osteoblasts was done by staining them for alkaline phosphatase using Sigma Diagnostics Procedure No. 85, where only osteoblasts stained blue (Rifas et al. 1989). The cell lines had been derived in our laboratory. Both cell lines were plated on three 60 x 15 mm diameter culture flasks (Nunclon®, Dern Intermed, Denmark). The culture medium used was Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies LTD, Paisley, Scotland) containing 5 % new-born calf serum, 1 % ascorbine (Merck, Darmstadt, Germany), 100 IU/ml penicillin, 100 g/ml streptomycin, 1 % L-glutamine and 0,1 % amphotericine B (Fungizone®, Life Technologies LTD, Paisley, Scotland). The cultures were maintained at 37 ºC temperature in an atmosphere of humidified air and 5 % carbon dioxide. They were passaged routinely with medium changes every second day and allowed to reach confluence before subculturing. Cells between the 6th and 10th passages were used. Cells were removed from the three flasks with trypsin (Life Technologies LTD, Paisley, Scotland). They were collected into one plastic tube and counted. Equal amounts of cells (1x 10⁵ cells) were then seeded on 40 mm diameter test flasks, each containing one test disc applied to the middle of the plastic flask and glued to the bottom by using sterile white soft paraffin. There were also control flasks without test discs and flasks with only sterile white soft paraffin of the same size as the test discs. Each flask contained 1 ml of culture medium, as described in detail above. The incubation conditions were also the same as above. The cells were then allowed to grow with medium changes every second day. At the end of the study, the cells were trypsinized and counted by using a hemocytometer. The total number of cell culture flasks was 18 for fibroblasts and 18 for osteoblasts. The culture flasks were photographed after a week of incubation, using a Wild MPS 51 camera (Heerburg, Switzerland) and a photoautomation device MPS 45 with 108x magnification.